Rab37-mediated exocytosis of IL-6 skews M2 polarization and activates STAT3 signaling in macrophages to promote lung cancer progression

Abstract

Background: Recent studies have revealed that bidirectional interactions between tumor cells and immune cells in the tumor microenvironment contribute to tumor progression Our preliminary data showed that mice systemically lacking Rab37 (Rab37 KO) suppressed tumor growth of subcutaneously injected with Lewis lung carcinoma (LLC) cells Interestingly Rab37 KO mice displayed low infiltration of pro-tumor M2 macrophages in LLC allografts suggesting that Rab37 possesses pro-tumor functions in tumor microenvironment However the role of Rab37 small GTPase-mediated exocytosis in stromal macrophages remains unknown Purpose: This study aims to investigate whether Rab37-mediated exocytosis skews macrophages toward M2 polarization and explore the mechanism involved Results: Our quantitative RT-PCR (qRT-PCR) and flow cytometry data showed that bone marrow derived macrophages (BMDMs) from Rab37 KO mice suppressed M2 macrophages polarization Next we performed the cytokine/chemokine arrays to reveal Rab37-mediated cytokines using conditioned media of BMDMs derived from wild-type (WT) or Rab37 KO mice Interestingly among the differential cytokines/chemokines the secreted level of interleukin-6 (IL-6) correlated with Rab37 expression in BMDMs Vesicle isolation revealed that IL-6 enriched in Rab37-specific vesicles in RAW264 7 macrophage cell line ELISA and immunofluorescence (IF) images confirmed that Rab37 mediated IL-6 secretion in RAW264 7 cell in a GTPase nucleotide-dependent manner i e IL6 secretion was upregulated in RAW264 7 cells expressing Rab37WT and GTP-bound Rab37Q89L In addition our cDNA microarray data showed that BMDMs from Rab37 KO mice displayed increased IFN-I signaling pathways compared to those from WT BMDMs after LLC conditional medium treatment Gene Set Enrichment Analysis (GSEA) and pathway analysis demonstrated that IFN?/β genes and STAT1 downstream interferon stimulated genes (ISGs) were upregulated in Rab37 KO BMDMs qRT-PCR assays confirmed the increased expression of IFNβ and ISGs genes including IFIT3 IRF7 and Mx1 in BMDMs from Rab37 KO mice In contrast expression of these genes was decreased in Rab37 overexpressing RAW264 7 cells which led to increased expression of M2 marker genes Arg1 and Ym1 Indeed ELISA data showed that the level of IFN-β was decreased in Rab37WT and Rab37Q89L RAW264 7 cells Among type I IFN upstream transcription factors we validated that STAT3 inhibited STAT1/IRF3 nuclear translocalization and suppressed type I IFN-associated genes expression through Rab37 mediated IL-6 secretion by immunoblotting and IF image Conclusions: Our results uncover a novel role of Rab37-mediated IL-6 secretion in regulating type I IFN signaling to skew macrophage polarization toward M2 Therapeutic strategies targeting Rab37/IL-6 axis in stromal macrophages in tumor microenvironment is a testable approach in the future

Date of Award	2019
Original language	English
Supervisor	Yi-Ching Wang (Supervisor)